Trefoil factor 1 (TFF1) is a tumor suppressor gene that encodes a peptide belonging to the trefoil factor family of protease-resistant peptides. Although TFF1 expression is frequently lost in gastric carcinomas (GCs), the tumorigenic pathways that are affected have yet to be determined. The aim of the current study was to identify the mechanism(s) by which the TFF1 gene is regulated in gastric carcinogenesis. In this study, TFF1 was shown to be silenced or downregulated in gastric tumor tissue compared with matched non-cancerous tissue. In addition, human gastric cells weakly expressed TFF1. The hypermethylation status in the promoter CpG islands appeared to be correlated with TFF1 expression levels in gastric cell lines or specimen tissue. Further molecular analysis indicated that the CpG islands play a role in the promoter activity of the TFF1 gene. The expression of TFF1 and DNA methylation of its promoter affected cell proliferation and apoptosis. The expression of TFF1 in gastric cell lines was restored with a demethylating agent, 5-azacytidine. Low expression of TFF1 in gastric cell lines and cancer tissue is associated with TP 53. In conclusion, the current study demonstrates that DNA methylation is a key mechanism of silencing TFF1 expression in human gastric cells and TFF1 gene hypermethylation of the CpG islands is a potential biomarker for GC.